Bioethanol production from renewable feedstock by baker's yeast Saccharomyces cerevisiae has become an attractive alternative to fossil fuels. However, the availability of starch or sucrose based feedstock such as corn grain or sugar cane is expected to be insufficient to cover future worldwide needs for bioethanol (Gray et al., 2006. Bioethanol. Current Opinion Chemical Biology. 10(2):141-146). A foreseen solution is the utilization of lignocellulosic feedstocks, such as corn stover, wheat straw, sugar cane bagasse, wood, etc (Hahn-Hägerdal et al., 2006. Bioethanol—the fuel of tomorrow from the residues of today. Trends Biotechnol. 24(12):549-556). This requires overcoming new challenges associated with the utilization of these complex raw materials.
One of these challenges concerns the presence of inhibitory compounds such as small aliphatic low molecular weight acids, furan derivatives, carbonyl compounds and phenolics that are released during the pretreatment and hydrolysis of lignocellulosic raw materials (Almeida et al., 2007. Increased tolerance of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol, 82:340-349). Among these compounds, the presence of the furan derivative 5-hydroxymethylfurfural (HMF), that originates from the dehydration of hexoses, has been reported to result in reduced ethanol productivity during the fermentation of lignocellulosic hydrolysates by S. cerevisiae (Taherzadeh et al. 2000. Physiological effects of 5-hydroxymethyl furfural on Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 53(6):701-708.). When compared in equimolar amounts with furfural, another inhibitory furan derivative found in hydrolysates, both the volumetric ethanol productivity and the sugar consumption rate by the cells are lower with HMF (Larsson et al., 1999. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme Microbial Technology 24:151-159.). Therefore, either removing HMF and/or improving cellular HMF detoxification are crucial for an industrial fermentation process based on lignocellulosic feedstocks.
WO03072602 discloses a polypeptide having Serin in position 295. However, the polypeptide lacks a leucine in position 117.
Nilsson et al., Applied and Environmetal Microbiology, December 2005, vol 71, page 7866-7871 disclosed that cell extracts from an lignocellulose hydrolysaste tolerant strain TMB3000 displayed a previously unknown NADH-dependent HMF reducing activity, which was not present in the less tolerant strain CBS 8066.
An absolute requirement for the development of fermentation processes based on lignocellulosic feedstocks is the development and optimisation of micro-organisms which are tolerant against the inhibiting compounds mentioned above or strain that can utilise/degrade the inhibiting compounds. S. cerevisiae strains have been shown to reduce HMF and furfural (Larsson et al., 1999. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme Microbial Technology 24:151-159) to 2,5-dimethanol (2,5-bis-hydroxymethylfuran) and 2-furanmethanol respectively, however the reduction rate is low and strain dependent. Strain TMB3000 (Lindén et al., 1992. Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant. Applied Environmental Microbiology. 58(5):1661-1669) appears to be, so far, the most tolerant strain that can grow in undiluted wood hydrolysates (Brandberg et al., 2005. Continuous fermentation of undetoxified dilute acid lignocellulose hydrolysate by Saccharomyces cerevisiae ATCC 96581 using cell recirculation. Biotechnology progress, 21(4):1093-1101).